{Reference Type}: Journal Article
{Title}: Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA 5-Hydroxymethylcytosine.
{Author}: Zhao J;Yan J;Li J;Shi G;Su M;Liu C;Jia G;
{Journal}: Anal Chem
{Volume}: 96
{Issue}: 32
{Year}: 2024 Aug 13
{Factor}: 8.008
{DOI}: 10.1021/acs.analchem.4c02621
{Abstract}: Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).